Abstract Mechanisms that maintain cancer stem cells are crucial to tumor progression. ID proteins are essential to support stemness, tissue invasion and angiogenesis in malignant glioma and other tumor types. Among ID proteins, ID2 contributes to cancer hallmarks, promotes chemoresistance of neural tumors and is part of a gene signature that predicts poor outcome in patients with high-grade glioma. Hypoxia?Inducible Factors (HIFs), most notably HIF2?, are expressed in and required for maintenance of cancer stem cells. However, the pathways that are engaged by ID2 or drive HIF2? accumulation during tumor progression have remained unclear. In this proposal, we will follow on our most recent work that has reconstructed the molecular events linking activation of ID2 and elevation of HIF2? in cancer. Our work indicates that disruption of the VHL-Elongin C-Elongin B (VCB)-Cul2 complex by un-phosphorylated ID2-Thr-27 is an important mechanism of HIF2? stabilization in stem cells and glioma stem cells (GSCs) and that ID2 activity is restrained by prolyl hydroxylase 1 (PHD1)-induced DYRK1 kinase activation and ID2-Thr-27 phosphorylation under normoxic conditions. Interestingly an ID2-T27A mutant constitutively inactivates VCB-Cul2, elevates HIF? and maintains cancer stem cells. In the proposal, we will characterize biochemically and functionally PHD-DYRK1 activity, a new tumor suppressor pathway that operates by restraining the interfering effect of active ID2 on VCB-Cul2 ubiquitin ligase. In Aim 1 we will identify the ID2- pT27 phosphatase, a potential oncoprotein and elucidate the broad significance of Thr-27 phosphorylation for the ID2 interactome. The biological implication of the PHD1-DYRK1-ID2-HIF2? pathway for glioma progression will be explored in Aim 2. Using PHD1Flox mice we will mechanistically dissect the function of constitutive ID2 activation in the absence of PHD1 in vivo. We will also model VHL inactivation, HIF? stabilization and gliomagenesis by ID2-T27A mutation in an inducible mouse model harboring a knock-in allele of the mutant ID2. We will use this mouse to validate the biochemical and biological effects of ID2-T27A that converge on VHL and HIF? in vivo. The mouse model will also be used to determine whether ID2-T27A promotes glioma progression in cooperation with other tumor-specific mutations that are known to cooperate with ID2 in human cancer. Studies will address fundamental questions in cancer biology and greatly enhance the understanding of how hallmarks of glioma progression are effected by ID2. Finally, the significance of the ID2-VHL-HIF-??axis for the human disease will be tested in patient-derived GSCs and primary GBM.